Am J Nucl Med Mol Imaging 2018;8(1):32-40 www.ajnmmi.us /ISSN:2160-8407/ajnmmi0069681 Original Article Synthesis, radiolabelling, and evaluation of [11C]PB212 as a radioligand for imaging sigma-1 receptors using PET Francesco Spinelli1,2, Ahmed Haider2, Annamaria Toscano1, Maria Laura Pati1, Claudia Keller2, Francesco Berardi1, Nicola Antonio Colabufo1, Carmen Abate1, Simon M Ametamey2 1Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari Aldo Moro, 70125, Bari, Italy; 2Department of Chemistry and Applied Biosciences, Center for Radiopharmaceutical Sciences of ETH-PSI-USZ, Institute of Pharmaceutical Sciences, ETH Zurich, CH-8093, Zurich, Switzerland Received November 23, 2017; Accepted December 20, 2017; Epub February 5, 2018; Published February 15, 2018 Abstract: The Sigma-1 receptor (Sig-1R) has been described as a pluripotent modulator of distinct physiological functions and its involvement in various central and peripheral pathological disorders has been demonstrated. However, further investigations are required to understand the complex role of the Sig-1R as a molecular chaperon. A specific PET radioligand would provide a powerful tool in Sig-1R related studies. As part of our efforts to develop a Sig-1R PET radioligand that shows antagonistic properties, we investigated the suitability of 1-(4-(6-methoxynaph- thalen-1-yl)butyl)-4-methylpiperidine (designated PB212) for imaging Sig-1R. PB212 is a Sig-1R antagonist and ex- hibits subnanomolar affinity (Ki = 0.030 nM) towards Sig-1R as well as good to excellent selectivity over Sig-2R. The radiolabelling of [11C]PB212 was accomplished by O-methylation of the phenolic precursor using [11C]MeI. In vitro autoradiography with [11C]PB212 on WT and Sig-1R KO mouse brain tissues revealed high non-specific binding, however using rat spleen tissues from CD1 mice and Wistar rats, high specific binding was observed. The spleen is known to have a high expression of Sig-1R. In vivo PET experiments in Wistar rats also showed high accumulation of [11C]PB212 in the spleen. Injection of Sig-1R binding compounds, haloperidol (1 mg/kg) or fluspidine (1 mg/ kg) shortly before [11C]PB212 administration induced a drastic reduction of radiotracer accumulation, confirming the specificity of 11[ C]PB212 towards Sig-1R in the spleen. The results obtained herein indicate that although [11C] PB212 is not suitable for imaging Sig-1R in the brain, it is a promising candidate for the detection and quantification of Sig-1Rs in the periphery. Keywords: PB212, sigma-1 receptor, PET imaging Introduction non-opioid, non-phencyclidine, and non-dopa- mine receptor so that the term ‘opioid’ was The term sigma receptor was initially intro- removed from the definition of the sigma recep- duced by Martin and colleagues in 1976 to tor (Sig-R) [4]. To date, we know that the sigma describe the target of the psychotomimetic system consists of at least two different recep- benzomorphan SKF-10,047 (N-allylnormetazo- tor subtypes, Sigma-1 (Sig-1R) and Sigma-2 cine) [1]. This receptor was first included in the (Sig-2R), differing in molecular weight, localiza- opioid system (sigma opioid receptor) since the tion, ligand engagement as well as biological effects of SKF-10,047 were blocked by the opi- functions [5, 6]. oid antagonist naltrexone [1]. However, subse- quent studies revealed that the SKF-10,047 The Sig-1R was cloned in 1996, yielding a pro- binding site was poorly inhibited by naltrexone tein of 223 amino acids which exhibits no and that the psychotomimetic effects of this homology with any other known mammalian drug in dogs were not antagonized by naltrex- protein [7, 8]. Several non-related endogenous one pretreatment [2, 3]. Hence, the sigma opi- compounds, such as steroids (e.g., progester- oid receptor introduced by Martin emerged as a one, dehydroepiandrosterone sulfate, etc.), the Evaluation of [11C]PB212 as a radioligand for imaging sigma-1 receptors hallucinogen N,N-dimethyltryptamine, and sp- [22] assays. Almost all Sig-1R radiotracers de- hingosine have been proposed as endogenous veloped to date are agonists and as such com- ligands of Sig-1R. However, their affinities are parison of the data obtained from [11C]PB212 not sufficient to explicitly define their role as with Sig-1R agonists, such as [11C]SA4503, endogenous ligands [4, 9-12]. Furthermore, could potentially provide important information Sig-1R shows moderate to high affinity towards about Sig-1R physiology, given that agonists a wide spectrum of exogenous ligands, thereby and antagonists may bind to different states of covering several different structural classes the receptor. With this in mind, we radiolabelled and with distinct pharmacological effects (neu- PB212 with carbon-11 and assessed its suit- roleptics, antidepressants, antitussives, drugs ability as a PET radioligand for imaging Sig-1R for the treatment of neurodegenerative disor- [24]. ders, drugs of abuse, etc.) [9]. Sig-1Rs are wide- ly distributed in the human body, both in the Materials and methods central nervous system (CNS) [13, 14] and in the periphery (mainly in spleen, liver, lung, kid- Animal experiments were conducted in accor- ney, and adrenal gland) [6, 7, 14-17]. At the sub- dance with the Swiss Animal Welfare legislation cellular level, the Sig-1R is mainly localized at and were approved by the Veterinary Office of the endoplasmic reticulum, specifically at the the Canton Zurich. Male Wistar rats were pur- interface with the mitochondrion, but it is also chased from Charles River (Sulzfeld, Germany) found in the plasma membrane and in the and kept under standard conditions. nuclear envelope [9, 18]. The information avail- All chemicals, unless otherwise stated, were able on the physiology and the pharmacology purchased from Sigma Aldrich GmbH (Taufkir- of Sig-1Rs supports their classification as chen, Germany), ABCR GmbH (Karlsruhe, Ger- molecular chaperons, regulating the activity of many), Merck (Darmstadt, Germany), or Fluka several cellular proteins, such as receptors, ion (Buchs, Switzerland) and were used without channels, kinases, etc. [9, 18, 19]. further purification. Fluspidine was kindly pro- Taken together, the wide anatomical and sub- vided by Prof. Bernand Wünsch (Westfälische cellular distribution, the ability to interact with Wilhelms-Universität Münster, Germany). several different exogenous ligands, as well Analytical radio-HPLC was performed with a as the chaperon activity towards different cel- flow rate of 1 mL/min on an Agilent 1100 series lular targets render the Sig-1R a pluripotent system equipped with a Raytest Gabi Star modulator [18] involved in many physiological radiodetector (Agilent Technologies, Morges, pathways and, consequently, in many central Switzerland). Semi-preparative HPLC purifica- and peripheral pathological disorders (compre- tions were carried out using a reversed phase hensive reviews: Cobos et al. [9], Su et al. [18], column (Phenomenex Luna, 5 µm, 250 × 10 Maurice and Su [19], Nguyen et al. [20]). How- mm) under the following conditions: 50 mM ever, further investigations are crucial to under- ammonium formate in H O pH = 4.4 (solvent A), stand the complex role of Sig-1Rs both in physi- 2 MeCN (solvent B); isocratic, 45% B; flow rate: 5 ological and pathological conditions. Hence, mL/min. Molar activity was calculated by com- the development of specific radiotracers for paring ultraviolet peak intensity of the final for- PET imaging provides a powerful tool in this mulated product with calibration curve of cor- direction. In this study, we aim to evaluate responding non-radioactive standard of known PB212 (1-(4-(6-methoxynaphthalen-1-yl)butyl)- concentrations. 4-methylpiperidine) [21] as a potential PET imaging agent for Sig-1R. PB212 exhibits sub- Radiochemistry nanomolar affinity towards Sig-1R K( i = 0.030 11 nM) [21], good to excellent selectivities against [ C]CO2 was produced by proton bombardment Sig-2R (Ki = 17.9 nM) [21], emopamil binding of nitrogen gas fortified with 0.5% oxygen us- protein (EBP, Ki = 8.04 nM) [21], serotonin (Ki ing IBA Cyclone 18/9 cyclotron (18-MeV; IBA, 5-HT1A > 1000 nM, Ki 5-HT7 > 1000 nM), dopa- Ottignies-Louvain-la-Neuve, Belgium) applying 14 11 mine (Ki D2R > 1000 nM, Ki D3R > 1000 nM) the well established N(p,a) C nuclear reacti- and adrenergic receptors (IC50 α1 > 100 nM) on. In a first step, nickel-based catalytic reduc- 11 11 [22]. In addition, PB212 has shown antagonis- tion of [ C]CO2 yielded [ C]CH4 which was sub- tic properties both in in vitro [23] and in vivo sequently iodinated to give [11C]MeI. Subse- 33 Am J Nucl Med Mol Imaging 2018;8(1):32-40 Evaluation of [11C]PB212 as a radioligand for imaging sigma-1 receptors 11 11 Scheme 1. Synthesis of precursor 1 [25] and radiosynthesis of [ C]PB212. a) BBr3, dry CH2Cl2, 78°C→rt; b) [ C] CH3I, Cs2CO3, dry DMF. quently, [11C]MeI was bubbled into a reaction (each 5 s) on ice, air dried, and exposed to a mixture containing phenolic intermediate 1 (1 phosphor imager plate for a period of 30 min. mg) and cesium carbonate (6 mg) in DMF (0.4 The plate was scanned using a BAS5000 read- mL) and stirred at 90°C for 3 min. After dilution er (Fujifilm, Dielsdorf, CH). of the crude product with water (1.6 mL), purifi- cation was performed by semi-preparative In vivo PET/CT imaging HPLC. The collected radiotracer was diluted PET and CT scans were obtained with a Super with water (8 mL) and passed through a C18 Argus PET/CT tomograph (Sedecal, Madrid, cartridge (Waters, pre-conditioned with 5 mL Spain) after injection of [11C]PB212 (18.1-21.4 EtOH and 10 mL water). After washing of the MBq, 0.58-1.36 nmol/kg) into the tail of male cartridge with water (5 mL), the product was Wistar rats (357-389 g, n = 3) which were kept eluted with EtOH (0.8 mL) into a sterile vial and under anesthesia using isoflurane.